我们要感谢宾夕法尼亚州立大学千禧科学综合体的流式细胞仪核心设施。

<ol>
	<li>Davies, L. C., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages:+Then+and+Now.">Tissue-resident macrophages: Then and Now.</a> Immunology. 144 (4), 541-548 (2015).</li><li>Kahn, S. E., Hull, R. L., Utzschneider, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+linking+obesity+to+insulin+resistance+and+type+2+diabetes.">Mechanisms linking obesity to insulin resistance and type 2 diabetes.</a> Nature. 444 (12), 840-846 (2006).</li><li>Van Gaal, L. F., Mertens, I. L., De Block, C. 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作者没有利益冲突的披露。

饮食诱导代谢紊乱模型, 模仿疾病, 如动脉粥样硬化, 简单的脂肪, 脂肪性肝炎和2型糖尿病广泛使用, 以更好地了解潜在的分子机制的疾病进展。胶原酶依赖性消化经常被用于游离组织, 从细胞外基质 (ECM)16,27中释放出癌细胞。酶, 如胶原蛋白酶扰乱胶原蛋白, 为相邻细胞提供结构支持。组织的结构组成决定了组织基质的刚度 (抵抗变形), 哪些粗胶原酶产品?...

小鼠模型已被广泛用于研究人类疾病的动态。从患病状态的小鼠中正确分离组织驻留细胞可以提供一个平台, 以了解分子和细胞对疾病发病机制的贡献1。肥胖是一种极其重要的疾病。随着胰岛素抵抗和2型糖尿病、心血管疾病和脂肪性肝病的发生, 肥胖的发病率在全球范围内持续上升2,3。通过减少脂肪组织产生的改变信号的体力活动而进一步扭曲了过量的养分消耗, 从而改变了其他外围组织的细胞环境, 如主动脉和肝脏4。这种扰乱代谢稳态导致慢性低等级系统性炎症5。
经典活化的巨噬细胞的主动脉和肝脏, 以及他们的招募白脂肪组织 (笏) 已被证明不仅启动失调的代谢信号, 但也持续炎症6,7。巨噬细胞表型和功能性异质性与肥胖相关疾病的发病机制有密切关系7。巨噬细胞极化的动态可塑性允许这些单元表现出一系列激活的表型, 以协调炎症的进展和分辨率8。虽然经典激活 (M1) 巨噬细胞是牵连炎症的传播, 或者激活 (M2) 巨噬细胞已与分辨率和组织修复相关的9,10。
当机体经历新陈代谢的压力时, 白色脂肪组织异常堆积。膨胀的脂肪组织吸引并保留了深刻改变正常脂肪细胞功能的炎性神经细胞, 以促进胰岛素抵抗、高血糖和最终2型糖尿病、胰岛素抵抗或高血糖11, 12。在平行, 白色脂肪组织改造反应的炎症信号的渗透经典活化 (M1) 脂肪组织巨噬细胞 (atm)13,14。这多器官施加一连串的信号, 出轨正常功能的其他机构, 如主动脉和肝脏4。
肝脏是一个新陈代谢的发电站, 适应的刺激来自附近的失调笏15。肝巨噬细胞或枯否体细胞, 在反应代谢变化, 分泌炎症细胞因子, 改变实质和实质细胞表型和促进组织重塑。肝脂堆积、炎症、细胞外基质沉积、坏死及最终功能丧失伴随着非酒精性脂肪肝引起的广泛肝损害的炎症反应16,17,18。
当身体承受慢性代谢压力19时, 大动脉在动脉壁内积聚脂质, 同时与受损的笏和肝功能相同。动脉脂质积累引发了活化的内皮细胞分泌的趋化因子, 并随后招募单核20。一旦被吸收, 单核细胞就会增殖, 分化, 摄取脂蛋白, 成为泡沫。动脉粥样硬化的启动和持续的炎活动的招募和组织居民脂质巨噬细胞。这些巨噬细胞屈服于动脉粥样硬化微环境中的胞外和胞内压力信号, 然后参与凋亡信号级联。由于这些泡沫细胞死亡, 他们贡献了他们的脂质填充内容的坏死核心病变, 然后导致斑块破裂, 心肌梗塞, 中风。
整体而言, 巨噬细胞表型的异质性协调在失调组织中观察到的炎症性改变引起的肥胖, 如笏、肝和主动脉8,21。特征的招募和组织驻留巨噬细胞可以提供洞察的潜在分子目标, 操纵巨噬细胞表型1。为了有效地描述由肥胖引起的炎症组织的巨噬细胞, 可以通过酶消化得到单细胞悬浮液。这种离解协议必须有效地在充分降解结缔组织, 同时尽量减少免疫细胞死亡和提供最佳的细胞产量。酶的混合物取决于组织的类型和它的结构构成。弹性组织, 如主动脉, 需要更强的酶活性, 比肝脏和笏, 以实现组织离解。从单细胞悬浮, 组织驻留巨噬细胞可以明确的特征或孤立的进一步的下游分析, 如转录剖面。
在这里, 一个组织特异性的协议描述, 使用胶原酶相关的组织消化和多色流式细胞仪有效分离和表征组织驻留巨噬细胞从传统的饮食诱发肥胖,动脉粥样硬化, 简单的脂肪和脂肪小鼠模型。同时染色细胞表面标记抗体抗白细胞-(CD45 和/或 CD11b) 和巨噬细胞 (F4/80) 特异抗原通常用于识别巨噬细胞的数量22。荧光活化细胞分类 () 是一种强有力的策略, 用来对这些被识别的种群进行高纯度的分类。然后利用下游分子分析 (如定量实时聚合酶链反应)23对表型特定基因图谱进行排序。虽然标准流式细胞仪和流 cytometry-based 细胞排序是强大的工具, 区分巨噬细胞在一个巨大的异构电池悬浮, 前协议必须优化, 以确保成功的输出。在本研究中, 描述了有效分离和表征可行组织特异性巨噬细胞的协议;更重要的是, 这项研究提供了对经常出现的技术问题的重要洞察力, 以及预防和/或解决它们的主动和 trouble-shooting 战略。

<table><tbody><tr><td>26 G x 5/8 in Needles</td><td>BD</td><td>305115</td><td></td></tr><tr><td>23 G x 0.75 in needle x 12 in. tubing Blood Collection Set</td><td>BD</td><td>367297</td><td>Used for cannulation of subhepatic IVC during liver perfusion</td></tr><tr><td>21 G 1 1/2 in. Needles</td><td>BD</td><td>305156</td><td></td></tr><tr><td>1 mL syringe with rubber stops</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10 mL Syringes</td><td>BD</td><td>309604</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>F4/80 PE</td><td>Biolegend</td><td>123110</td><td></td></tr><tr><td>CD11c PE/Cy7</td><td>Biolegend</td><td>117318</td><td></td></tr><tr><td>CD11b PE/Cy5</td><td>eBioscience</td><td>15-0112-81</td><td></td></tr><tr><td>Anti-mouse CD16/32 Fc Block&amp;nbsp;</td><td>Biolegend</td><td>101320</td><td></td></tr><tr><td>CD45 Pacific Blue</td><td>Biolegend</td><td>103126</td><td></td></tr><tr><td>PE Rat IgG2a</td><td>Biolegend</td><td>400508</td><td></td></tr><tr><td>PE/Cy7 Armenian Hamster IgG</td><td>Biolegend</td><td>400922</td><td></td></tr><tr><td>PE/Cy5 Rat IgG2b</td><td>Biolegend</td><td>400610</td><td></td></tr><tr><td>Pacific Blue Rat IgG2c</td><td>Biolegend</td><td>400717</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Cellgro</td><td>15-017-CV</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS)</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>70 &amp;mu;m cell strainers</td><td>Corning, Inc.</td><td>352350</td><td></td></tr><tr><td>1.7 mL microcentrifuge tube&amp;nbsp;</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Paraformaldehyde Aqueous Solution -16x</td><td>Electron Microscopy Sciences</td><td>CAS #30525-89-4</td><td></td></tr><tr><td>Micro Dissecting Scissors, 3.5 inch, Straight, 23 mm, Sharp&amp;nbsp;</td><td>Stoelting</td><td>52132-10P</td><td>Used for general dissecting purposes</td></tr><tr><td>Micro Forceps, 4 inches, full curve, 0.8 mm</td><td>Stoelting</td><td>52102-37P&amp;nbsp;</td><td>Used for general dissecting purposes</td></tr><tr><td>Spring dissection scissors &amp;ndash; 3 mm Cutting edge&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td></tr><tr><td>Curved 0.07 mm x 0.04&amp;nbsp;mm Tip Forceps</td><td>Fine Science Tool</td><td>11297-10</td><td>Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td></tr><tr><td>Hemostatic Forceps (Curved)</td><td>Fine Science Tool</td><td>13021-12</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Fischer Scientific</td><td>9041-08-1</td><td></td></tr><tr><td>35 mm Cell Culture/Petri Dishes</td><td>Fischer Scientific</td><td>12-565-90</td><td></td></tr><tr><td>Polystyrene Petri Dishes (10 cm) w/lid</td><td>Fischer Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>15 mL Conical Centrifuge Tubes (Polypropylene)</td><td>Fischer Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes&amp;nbsp;(Polypropylene)</td><td>Fischer Scientific</td><td>14-432-22</td><td></td></tr><tr><td>5 mL Round-Bottom Polystyrene Tubes&amp;nbsp;</td><td>Fischer Scientific</td><td>14-959-5</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td><td>Millipore</td><td>EX0285-1</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Rockland</td><td>BSA-50</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Collagenase Type II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>Collagenasse Type XI</td><td>Sigma-Aldrich</td><td>C7657</td><td></td></tr><tr><td>Hyaluronidase Type I</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Collagenase Type I</td><td>Sigma-Aldrich</td><td>C0130</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>1310-73-2&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>449709-10G</td><td></td></tr><tr><td>500 mL beaker&amp;nbsp;</td><td>Sigma Aldrich</td><td>02-540M</td><td></td></tr><tr><td>4 cm Hemostatic clamp</td><td>Stoelting</td><td>52120-40</td><td></td></tr><tr><td>Toothed forceps</td><td>Stoelting</td><td>52104-33P</td><td></td></tr><tr><td>50 &amp;mu;m Disposable filters</td><td>Systemex</td><td>04-0042-2317</td><td></td></tr><tr><td>Collagenase Type IV</td><td>ThermoFischer Scientific</td><td>17104019</td><td></td></tr><tr><td>Ammonium Chloride Potassium (ACK)</td><td>ThermoFischer Scientific&amp;nbsp;</td><td>A1049201</td><td></td></tr><tr><td>Razors (0.22 mm (0.009&quot;))</td><td>VWR International</td><td>55411-050</td><td></td></tr><tr><td>Texas Red Live/Dead stain</td><td></td><td></td><td>Red viability stain (in &lt;strong&gt;Figure 1A&lt;/strong&gt;)</td></tr></tbody></table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

肥胖促进了一种慢性炎症状态, 主要由组织驻留的巨噬细胞以及单核细胞衍生的巨噬病介导。饮食诱发肥胖 (迪奥) 是研究巨噬细胞异质性作用的有价值的模型;然而, 足够的巨噬细胞隔离很难从发炎的组织中获得。在本协议中, 我们概述了从我们的研究获得一个合适的人群组织驻留巨噬细胞后18周高脂肪 (风) 或高脂肪/胆固醇的隔离步骤和必要的故障排除准则 (HFHCD) 饮食干预。本议定书的重点是三标志性组织研究的肥胖和动脉粥样硬化, 包括肝脏, 白脂肪组织 (笏), 和主动脉。我们强调流式细胞术的二元用法如何实现组织驻留巨噬细胞的分离和表征的新维度。本议定书的一个基本部分解决复杂的基础组织特定的酶消解和巨噬细胞分离, 以及随后的细胞表面抗体染色流细胞分析。这项协议解决了现有的复杂的荧光活化细胞分类 (资产管制), 并提出了这些复杂性的澄清, 以获得广泛的范围, 从适当的排序细胞群体的描述。可选的浓缩方法包括对细胞进行分类, 例如稠密的肝脏, 允许在与外地资产管理体系合作时具有灵活性和时间性。简言之, 这项协议帮助研究者评估了一项研究中大量炎症组织的巨噬细胞异质性, 并提供了有洞察力的诊断技巧, 以利于细胞的分离和鉴定。免疫细胞在迪奥介导的炎症。

当使用载脂蛋白 E 缺乏 (载脂蛋白 KO) C57BL/6 (BL6) 小鼠维持高脂肪高胆固醇饮食 (HCHFD 或 HCD) 18 周, 1 x 104 -2 x 104 CD45+F4/80+主巨噬细胞可以被隔离当两个样本汇集.肝解剖从 HFHCD 喂食的载脂蛋白高鼠, 产生大于 5 x 105排序的枯否细胞 (这取决于可用的排序时间)。当使用高脂肪饮食 (风) 喂养野生型 (重量级) C57BL/6 小鼠, 5 x 105到 1 x 10...

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

该协议允许研究员分离和表征组织驻留巨噬细胞在各种标志发炎组织提取从饮食诱发的代谢紊乱模型。

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

isolation-characterization-purification-macrophages-from-tissues

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

25.1K 次观看.  The Pennsylvania State University. 该协议允许研究员分离和表征组织驻留巨噬细胞在各种标志发炎组织提取从饮食诱发的代谢紊乱模型。

视频: Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

生物化学

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

体外骨髓源性巨噬细胞对髓鞘碎片的吞噬

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

神经科学

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

医学

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...